Cyclic peptide analogs of melanocortin and amanitin and methods of making such

ABSTRACT

The invention described herein is based in part on the discovery of a protein/peptide crosslink, which introduces fluorescent properties, and which has been applied to synthesize analogues of melanocortin and amanitin as choice peptides to be explored in the context of isoindole peptides. Without limitation, it is expected that those trained in the art of peptide synthesis and stapling would appreciate the consequences of this invention such that other peptides of varied length can be similarly constrained by isoindole staples as featured herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/804,465, filed on Feb. 28, 2020, which claims priority to U.S. Provisional Patent Application No. 62/812,410 filed Mar. 1, 2019, each disclosure is hereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: A9TH_014_02US_SeqList_ST25.txt, date recorded: Aug. 9, 2023, file size 5,256 bytes).

TECHNICAL FIELD

This invention relates to methods of cyclization of biologicals, such as peptides (e.g. melanocortins and amatoxins), using a cross-linking approach. In particular, the invention relates to linear and cyclic derivatives of the naturally occurring peptide alpha-melanocyte stimulating hormone (alpha-MSH), which has application in therapy, imaging, and targeted therapy, and to bicyclic peptide analogs of amanitin with florescent properties.

BACKGROUND

The pursuit of secondary structure stabilization is an ubiquitous goal in peptide chemistry and of central importance to the utilization of peptides as medicinal probes and high affinity ligands¹. To this end, Nature typically employs oxidative disulfide formation from two cysteine thiols². Disulfide crosslinks are also exploited in the development of synthetic peptides as drugs and imaging agents³. Since disulfide linkages are labile under reducing conditions, Nature uses redox-inert crosslinks in natural products, most notably, thioethers, as seen in the lanthionine antibiotics⁴, tryptathionine linkages as seen amatoxins and phallotoxins⁵, and heteroaryl crosslinks between tryptophan and histidine as seen in celogentin⁶, in addition to several that have been recently reviewed⁷. These chemical crosslinks are critical to the biological activity of these molecules, whose syntheses are of enduring interest⁸. In considering the potency of these peptide natural products, the development of facile, synthetic crosslinking methodologies continues to be an active field of study. Such strategies offer the potential for truncating longer natural peptide sequences, thus facilitating scalable synthesis and improving pharmacokinetics⁹. More importantly, crosslinks rigidify the scaffold by reducing flexibility to favor a limited envelope of secondary structures. When applied properly, conformational restriction may dramatically improve both affinity and specificity^(9e, 10).

Towards these ends, many well-known reactions within the synthetic repertoire have been repurposed for novelty in peptide crosslinking as reviewed extensively¹¹; key examples include stapling by olefin cross-metathesis¹², Glaser alkyne synthesis¹³, Cu-catalyzed triazole formation¹⁴, oxadiazole formation¹⁵, a host of thioether-forming reactions¹⁶, and novel alkylation¹⁷ and arylation reactions¹⁸, all of which augment the many standard reactions made possible by commercially available homo- and heterobifunctional crosslinking reagents. Whereas rapidity, chemoselectivity, and high-yielding outcomes represent critical necessities of a given crosslinking method, they are not sufficient to guarantee high affinity (specific examples of crosslinked peptides with comparatively elevated K_(d)s include analogs of octreotatel^(14c) and α-MSH¹⁹). Moreover, few crosslinking methods afford the added advantage of an emergent fluorescent signature as a direct consequence of crosslinking to provide spectroscopic evidence of successful reaction. Therefore, new crosslinking methods are needed that not only increase stability of the secondary peptide structure but also introduce useful features, such as fluorescence, to the structure.

DESCRIPTION

Inspired by certain indole-derived crosslinks found in highly strained peptide natural products, we sought such a facile and highly chemoselective crosslinking reaction for generating bioactive mono- and bicyclic peptides that would be accompanied by the prospect of emergent fluorescence. An outstanding candidate reaction that simultaneously meets these multiple criteria may be the condensation of an amine, a thiol, and ortho-phthalaldehyde (OPA) that may result in a highly fluorescent isoindole. Further advantages may include the use of mildly basic, transition metal-free, aqueous conditions that may be compatible with most biological molecules as well as high atom-economy.

Isoindole synthesis is known in other contexts; most commonly it is used analytically to quantify peptides of unknown extinction coefficients whereby free amines (N-terminus, lysines) are converted to highly fluorescent isoindoles in the presence of excess thiol²⁰. Ortho-phthalaldehyde has also been used in mechanistic enzymology as an active-site titrant²¹ and as an affinity label for studying steroid and opioid receptors²² and kinases²³. Yet apart from a few isolated reports on isoindole crosslinking of glutathione for the sake of analytical/mechanistic applications²⁴, the use of OPA to drive crosslinking between a side-chain amine and a cysteine with concomitant fluorescent isoindole formation (FIG. 1 ) to form mono- and bicyclic peptides remains undocumented in the vast literature of peptide crosslinking reactions.

While it would be obvious to those trained in the art of peptide synthesis and stapling to consider this application in the context of any number of peptide lengths (e.g. peptides of 4-20 amino acids in length) to test this application, we sought a clinically relevant peptide known to require a β-turn motif for high-affinity target binding. Alpha-melanocyte stimulating hormone (α-MSH) provided an ideal test case: its interactions with melanocortin receptors require a specific β-turn motif that has been extensively exploited in the context of linear and monocyclic peptides ranging from 4-10 amino acids²⁵. High-affinity α-MSH derivatives are of considerable interest for imaging²⁶ and treating melanoma²⁷, based on targeting the melanocortin-1 receptor (MC1-R) that is specifically expressed in the majority of melanomas.

Hence, we began our investigation by preparing four linear heptapeptide precursors containing a single cysteine to address different lengths of the amine. All were cyclized in a single, rapid, high-yielding step to provide new MSH analogs with fluorescent isoindole crosslinks that show high affinity denoted by low (single-digit) nanomolar dissociation constants. Because isoindole condensation remains an unexplored area of study, we synthesized five additional OPAs to access a portfolio of corresponding isoindoles with altered photophysical properties and increased photostability, all of which are installed late stage within 15 min. Finally, we applied the same strategy for intra-annular crosslinking of an octapeptide macrocycle to create a bicycle; in a preliminary test, we prepared an analog of α-amanitin wherein the isoindole replaces the characteristic tryptathionine crosslink. Surprisingly, this new bicyclic product showed detectable cytotoxicity that is further understood with corroborating molecular modelling. In light of the ease, rapidity, chemoselectivity, and robustness of this reaction, we have named this approach, “FlICk-ing”—fluorescent iso-indole crosslinking. Based on these promising results, we posit that this work will have broad applications to peptide and protein chemistry (vide infra).

Furthermore, the resulting crosslinked peptide may be endowed with both rigidity and the added value of fluorescence in the context of a highly biologically active and medicinally relevant peptide sequence. Additionally beneficial may be the fact that the fluorophore is incorporated into the main structure of the peptide, instead of appended onto one of the termini or onto a side chain. Thus implying that the structure of the peptide arrived at through discovery may be already the structure of a viable fluorescent probe. This obviates the need for post sequence discovery/optimization fluorescent derivatization, which can often negatively impact the binding affinity. Employing fluorescent isoindole crosslinking (FLIC) towards discovery and optimization may result in lead that require no further structural elaboration to synthesize probes.

SUMMARY

The invention described herein is based in part on the discovery of a protein/peptide crosslink, which introduces fluorescent properties, and which has been applied to synthesize analogues of melanocortin and amanitin as choice peptides to be explored in the context of isoindole peptides. Without limitation, it is expected that those trained in the art of peptide synthesis and stapling would appreciate the consequences of this invention such that other peptides of varied length can be similarly constrained by isoindole staples as featured herein.

In one embodiment the invention described herein provides a method of creating an intramolecular crosslinking within a peptide comprising: (a) Providing a peptide composition that further comprises at least one thiol group and at least one amine group and (b) reacting the peptide with a crosslinking agent of Formula VIII.

-   -   Wherein:     -   A², A³, A⁴ and A⁵ may be hydrogen, halogen, NO₂. CN, alkyne,         azide, aryl, heteroaryl, COOH, SO₃ ⁻, CF₃, RCO, CONHR, NH₂, NHR,         NR₂, ether, thioether, hydroxyl, or boronate; wherein R may be         an alkyl or alkenyl, optionally substituted; and     -   B², B³, B⁴ and B⁵ may be carbon or nitrogen.

In one embodiment the present invention comprises a linear analogue of alpha-MSH of Formula I R¹-R²—R*—X¹—X²—X³—X⁴—R³-R⁴  (Formula I);

-   -   wherein R¹ optionally may —H or —Ac;         -   R² may be -Nle-;         -   X¹ may be -His- or -D-His-;         -   X² may be -Phe- or -D-Phe-;         -   X³ may be -Arg- or -D-Arg-;         -   X⁴ may be -Trp- or -D-Trp-;         -   R³ may be -Cys- or -D-Cys-;         -   R⁴ optionally may be —H or —NH₂;         -   R* may be —NH—CH(R^(#)NH₂)—C(═O)—NH₂; and wherein R^(#) may             be a linear and optionally substituted alkyl.

In another embodiment the present invention comprises a linear analogue of alpha-MSH, wherein R* may be

with m=1-15.

In another embodiment the present invention comprises a cyclic analogue of alpha-MSH of Formula IIa

In another embodiment the present invention comprises a cyclic analogue of alpha-MSH of Formula IIb

-   -   Wherein R¹ optionally may be Ac— or H—;     -   R² may be -Nle-;     -   X¹ may be -His- or -D-His-;     -   X² may be -Phe- or -D-Phe-;     -   X³ may be -Arg- or -D-Arg-;     -   X⁴ may be -Trp- or -D-Trp-;     -   A¹⁻⁵ may be hydrogen, halogen, NO₂. CN, alkyne, azide, aryl,         heteroaryl, SO₃ ⁻, CF₃, RCO, COOH, CONHR, NH₂, NHR, NR₂, ether,         thioether, hydroxyl, or boronate; wherein R may be an alkyl or         alkenyl, optionally substituted;     -   B¹⁻⁵ may be carbon or nitrogen; and     -   x and y independently may be a natural number between 1-15.

In another embodiment the present invention comprises a cyclic analogue of alpha-MSH of Formula IIc:

-   -   wherein variables may be defined as above.

In a further embodiment the present invention comprises a cyclic analogue of alpha-MSH of Formula III

In one embodiment the present invention comprises the following cyclic analogues of alpha-MSH (Formula IV a-d):

In a further embodiment the present invention comprises a pharmaceutical formulation of one or more compounds of Formula I-IV.

In a further embodiment the present invention comprises a cosmetic formulation of one or more compounds of Formula I-IV. Wherein the cosmetic formulation may further comprise a cosmetically-acceptable carrier or diluent.

In one embodiment the present invention comprises an amatoxin of Formula V:

-   -   wherein     -   R′¹ may be NH₂, OH, NHR′, NHOR′, or NHNHR′;     -   R′² may be H, OH, OR′, ═O, NH₂, SH, NHR′, N₃, NH(C═NH₂ ⁺)NH₂         i.e. guanidinium, or CN;     -   R′³ may be H, F, Cl, NH₂, N₃, ═O, SH, NHR′, OR′, SR′, or OH;     -   R′⁴ may be H, F, Cl, NH₂, N₃, ═O, SH, NHR′, OR′, SR′, or OH;     -   R′⁵ may be H, linear or cyclic alkyl or alkenyl, optionally         further substituted and/or comprising heteroatoms;     -   R′⁶, R′⁷, R′⁸, and R′⁹ independently may be H, D, or alkyl that         optionally further may be substituted and/or comprises         heteroatoms;     -   X^(a) and X^(b) independently may be (CH₂)_(n) with n=0-5, NH,         or NR′;     -   Z may be an isoindole;     -   R′ may be wherein R′ may be H, a linear or cyclic C₁₋₂₀ alkyl         chain that may be optionally substituted, an aryl or a         heterocycle that may be optionally substituted, a tertiary         amide, or a linker group suitable for biomolecule conjugation.

Examples of said isoindoles for Z may comprise

-   -   wherein     -   A′¹⁻⁵ may be hydrogen, halogen, NO₂. CN, alkyne, azide, aryl,         heteroaryl, SO₃ ⁻, CF₃, RCO, COOH, CONHR, NH₂, NHR, NR₂, ether,         thioether, hydroxyl, or boronate; wherein R may be an alkyl or         alkenyl, optionally substituted;     -   B′¹⁻⁵ may be carbon or nitrogen; and     -   o and p independently may be 0, 1, 2, 3, or 4.

In a further embodiment the present invention comprises an amatoxin of Formula IV, wherein Z may be

with o and p independently 0, 1, 2, 3, or 4.

In another embodiment the present invention comprises an amatoxin of Formula VI:

-   -   Wherein said amatoxin of Formula VI may be FlICk-amanitin-1.

In another embodiment the present invention comprises an amatoxin of Formula VII:

-   -   Wherein said amatoxin of Formula VII may be FlICk-amanitin-2.

In a further embodiment, the present invention comprises a pharmaceutical composition of any one or more compounds of Formula V-VII.

In one embodiment the present invention comprises the use of one or more compounds of Formula I-IV to increase alpha-MSH activity in a subject in need thereof.

In another embodiment the present invention comprises the use of one or more compounds of Formula I-IV for the manufacture of a medicament having melanotropic activity.

In a further embodiment the present invention comprises the use of one or more compounds of Formula I-IV as an inhibitor binding to melanocortin receptor 1 (MCR1).

In a further embodiment the present invention comprises the diagnostic use of one or more compounds of Formula I-IV. For example, as medical imaging agents.

In a further embodiment the present invention comprises the use of one or more compounds of Formula I-IV for the diagnosis and/or treatment of neurodegenerative diseases. Wherein examples of neurodegenerative diseases may comprises multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), dementia, Alzheimer's disease, Parkinson's disease, and Huntington's disease.

In a further embodiment the present invention comprises the use of one or more compounds of Formula I-IV for the treatment of skin associated diseases. Wherein examples of skin associated diseases may comprise photosensitivity, photodermatoses, psoriasis, bullous disease, and Hailey-Hailey disease.

In a further embodiment, the present invention comprises a method of use of any one or more of the compounds of Formula V-VII for use in the treatment of cancer in a subject in need thereof.

In one embodiment the present invention provides a method of cyclizing a peptide by strategically crosslinking a thiol group with an amine group, said method comprising:

-   -   (i) preparing a linear peptide of Formula I;     -   (ii) crosslinking said linear peptide of Formula I with a         cross-linking agents to give a cyclized peptide of Formula III;         and     -   (iii) optionally isolation and/or further reacting said cyclized         peptide formed in step (ii).     -   Wherein said cross-linking agent may be a substituted aromatic         compound, a fluorescent compound, a dye,     -   Wherein said cross-linking agent may be a compound of Formula         VIII:

-   -   A²⁻⁵ may be hydrogen, halogen, NO₂. CN, alkyne, azide, aryl,         heteroaryl, SO₃ ⁻, CF₃, RCO, COOH, CONHR, NH₂, NHR, NR₂, ether,         thioether, hydroxyl, or boronate; wherein R may be an alkyl or         alkenyl, optionally substituted; and     -   B²⁻⁵ may be carbon or nitrogen.

In one example said cross-linking agent may be specifically:

In one embodiment the present invention provides a method of cyclizing a peptide by strategically crosslinking a thiol group with an amine group, said method comprising:

-   -   (i) making a solid phase compatible version of a heptapeptide;     -   (ii) cleaving said heptapeptide from step (i) and coupling it         with dihydroxyisoleucine;     -   (iii) performing isoindole condensation to obtain a monocycle;     -   (iv) macrolactamizing said octapeptide to provide said compound         of Formula V; and     -   (v) optionally deuterating said compound of Formula V.     -   Wherein steps (iii) and (iv) may be conducted in reverse order.     -   Wherein said heptapeptide may comprise diaminoproprionic acids.         For example, wherein said heptapeptide may be

-   -   Wherein said deuterated version of said compound of Formula V         from step (v) may be more metabolically stable.

Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprising” and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of “including, but not limited to”

DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings.

FIG. 1 : A: standard termolecular condensation to give an isoindole to quantify the concentration of free amine in a peptide or biological sample; B: isoindole crosslinking on a linear peptide to give a macrocycle; C: isoindole crosslinking on a peptide macrocycle to give a bicyclic structure.

FIG. 2 : At left: structure of Melanotan-II; At right: rapid, high yielding FlICk synthesis of α-MSH derivatives with different ring sizes.

FIG. 3 : Modified OPAs used in this study.

FIG. 4 : At left: α/β-amanitins; middle: FlICk-amanitin-1; At right: FlICk-amanitin-2; each bicycle has the same overall connectivity of 5 intervening atoms in either the tryptathionine or isoindole crosslink.

FIG. 5 : Synthesis of the FlICk-amanitin-1.

FIG. 6 : Cytotoxicity assays—CHO cells were incubated with α-amanitin, the monocyclic octapeptide precursor and the FlICk-amanitin-1 for 72 h in triplicate and cell viability was assessed by spectrophotometry with MTT indicator.

FIG. 7 : Synthesis of Substituted o-Benzylic Diols. Permangate oxidation of substituted o-xlyenes under mild aqueous conditions followed by reduction of the resulting phthalic acids with BH₃-THF complex.

FIG. 8 : Synthesis of Substituted o-Phthalaldehydes. Swern oxidation of o-benzylic diols yields o-phthalaldehyde derivatives that were isolated as methyl-hemiacetals due to their incompatibility with aqueous workup.

FIG. 9 : Synthesis of Isoindole α-Amanitin Derivative. On resin synthesis of a linear octapeptide followed by mild acid cleavage to leave the protecting groups intact. Subsequent elaboration to a full length octapeptide followed by macrolactamization and global deprotection sets the stage for the ultimate step; intra-annular isoindole condensation. Synthesis of the protect dihydroxyisoleucine moiety was detailed in our previous report on the total synthesis of α-amanitin¹⁰.

FIGS. 10A-10D: HPLC chromatographs for purified product reinjection. (A) linear octapeptide, 219 nm, H₂N-DhIle(Bis-O-TBS)-Dap(Boc)-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(OtBu)-OH, 219 nm channel; (B) FlICk-amanitin, 230 nm channel; (C) FlICk-amanitin, 260 nm channel; (D) FlICk-amanitin, 335 nm channel.

FIGS. 11A-11B: MTT cell viability assay against CHO cells: (A) alpha-amanitin; (B) FlICk-amanitin.

FIG. 12 : Sketch illustrating the underlying crosslinking concept.

DETAILED DESCRIPTION

Inspired by certain indole-derived crosslinks found in highly strained peptide natural products, we sought such a facile and highly chemoselective crosslinking reaction for generating bioactive mono- and bicyclic peptides that would be accompanied by the prospect of emergent fluorescence. An outstanding candidate reaction that simultaneously meets these multiple criteria may be the condensation of an amine, a thiol, and ortho-phthalaldehyde (OPA) that may result in a highly fluorescent isoindole. Further advantages may include the use of mildly basic, transition metal-free, aqueous conditions that may be compatible with most biological molecules as well as high atom-economy.

Isoindole synthesis is known in other contexts; most commonly it is used analytically to quantify peptides of unknown extinction coefficients whereby free amines (N-terminus, lysines) are converted to highly fluorescent isoindoles in the presence of excess thiol²⁰. Ortho-phthalaldehyde has also been used in mechanistic enzymology as an active-site titrant²¹ and as an affinity label for studying steroid and opioid receptors²² and kinases²³. Yet apart from a few isolated reports on isoindole crosslinking of glutathione for the sake of analytical/mechanistic applications²⁴, the use of OPA to drive crosslinking between a side-chain amine and a cysteine with concomitant fluorescent isoindole formation (FIG. 1 ) to form mono- and bicyclic peptides remains undocumented in the vast literature of peptide crosslinking reactions.

To test this application, we sought a clinically relevant peptide known to require a β-turn motif for high-affinity target binding. Alpha-melanocyte stimulating hormone (α-MSH) provided an ideal test case: its interactions with melanocortin receptors require a specific β-turn motif that has been extensively exploited in the context of linear and monocyclic peptides ranging from 4-10 amino acids²⁵. High-affinity α-MSH derivatives are of considerable interest for imaging²⁶ and treating melanoma²⁷, based on targeting the melanocortin-1 receptor (MC1-R) that is specifically expressed in the majority of melanomas.

Hence, we began our investigation by preparing four linear heptapeptide precursors containing a single cysteine to address different lengths of the amine. All were cyclized in a single, rapid, high-yielding step to provide new MSH analogs with fluorescent isoindole crosslinks that show high affinity denoted by low (single-digit) nanomolar dissociation constants. Because isoindole condensation remains an unexplored area of study, we synthesized five additional OPAs to access a portfolio of corresponding isoindoles with altered photophysical properties and increased photostability, all of which are installed late stage within 15 min. Finally, we applied the same strategy for intra-annular crosslinking of an octapeptide macrocycle to create a bicycle; in a preliminary test, we prepared an analog of α-amanitin wherein the isoindole replaces the characteristic tryptathionine crosslink. Surprisingly, this new bicyclic product showed detectable cytotoxicity that is further understood with corroborating molecular modelling. In light of the ease, rapidity, chemoselectivity, and robustness of this reaction, we have named this approach, “FlICk-ing”—fluorescent iso-indole crosslinking. This work will have broad applications to peptide and protein chemistry (vide infra).

Furthermore, the resulting crosslinked peptide may be endowed with both rigidity and the added value of fluorescence in the context of a highly biologically active and medicinally relevant peptide sequence. Additionally beneficial may be the fact that the fluorophore is incorporated into the main structure of the peptide, instead of appended onto one of the termini or onto a side chain. Thus implying that the structure of the peptide arrived at through discovery may be already the structure of a viable fluorescent probe. This obviates the need for post sequence discovery/optimization fluorescent derivatization, which can often negatively impact the binding affinity. Employing fluorescent isoindole crosslinking (FLIC) towards discovery and optimization may result in lead that require no further structural elaboration to synthesize probes.

Materials and Methods

The procedures described herein are given for the purposes of example and illustration only and should not be considered to limit the spirit or scope of the invention.

General Peptide Synthesis Procedure

Unless otherwise indicated, all solvents and reagents were purchased from Sigma-Aldrich, Fischer Scientific, Alfa-Aesar and Cambridge Isotope Laboratories. Purchased products were used without further purification. Dry THE was obtained from distillation and stored with 4 Å molecular sieves, while other dry solvents such as DMF, DCM, EtOAc, and MeCN were dried over and stored with 4 Å molecular sieves at least one day prior to use. Columns used for purification were packed using SiliaFlash F60 (230-400 mesh silica gel from Silicycle, unless otherwise stated, in which case Merck Grade Silica purchased from Sigma Aldrich was used. Low resolution mass spectrometry data were acquired using a Waters ZQ GC-MS coupled to a Waters 2695 HPLC for the purposes of sample injection. High resolution mass spectrometry data were taken by Derick Smith on a Waters/Micromass LCT TOF Mass Spectrometer. ¹H NMR, ¹³C NMR, ¹⁹F NMR, COSY, HSQC and HMBC spectroscopy data were collected on a 300 MHz Bruker Avance Spectrometer unless otherwise stated, in which case the data were collected on a 600 MHz Bruker Avance Spectrometer. All chemical shifts are reported in ppm (δ) scale, and are referenced to the solvent signal. HPLC purifications were performed using an Agilent 1100 Series paired with an Agilent Eclipse XDB-C18 column. Detection was through UV-Vis absorbance at 229 nm, 283 nm, 335 nm, or 450 nm depending on the product being eluted. Regular peptides were monitored at 283 nm due to tryptophan absorbance, isoindoles, 3-fluoro isoindoles and 4-fluoroisoindoles were measured at 335 nm, and 3 and 4 nitro isoindoles were detected at 450 nm.

O-Phthalaldehyde Derivatives

The methodology for the synthesis of all o-phthalaldehyde derivatives is largely the same as that for the sample 4-nitrophthalaldehyde synthesis below. Any details that differ are stated in the corresponding sections for the phthalaldehyde derivatives. Full spectra and characterization data can be found in appendix A.

4-Nitrophthalic Acid

1,2-dimethyl-4-nitrobenzene (5.02 g, 33.18 mmol) was added to a deep purple solution of 150 mL of milliQ water and potassium permanganate (KMNO₄, 43.25 g, 288.27 mmol) and sonicated overnight. The reaction flask was then removed from the sonicator and quenched at 0° C. using KOH pellets and EtOH until the contents of the flask were dark brown. The resulting mixture was filtered to remove solid by-products and acidified to pH ˜0 with concentrated HCl_((aq)). The acidified mixture was extracted into EtOAc (3×75 mL) and the organic layers were combined, dried with MgSO₄ and solvent was removed under reduced pressure. The resulting solid was used in the next step without purification. Yield: 3.03 g, 50%; ¹H NMR (300 MHz, CD₃CN) δ 8.53 (d, J=2.3 Hz, 1H), 8.40 (dd, J=8.5, 2.3 Hz, 1H), 7.90 (d, J=8.5 Hz, 1H). ¹³C NMR (75 MHz, CD₃CN) δ 167.65, 166.48, 149.96, 139.06, 133.45, 131.08, 127.34, 125.12, 118.26. ESI-HRMS for C₈H₅NO₆: [M+Na⁺]⁺: calculated: 234.0009. found: 234.0015.

(4-nitro-1,2-phenylene)dimethanol

4-nitrophthalic acid (3.68 mmol, 772.8 mg) was added to a previously flame dried flask containing a stir bar and dry THF (1 mL). The mixture was stirred until no solids were observed. 1 M BH₃·THF solution (18.5 mmol, 18.5 mL) was then slowly added to the mixture at −78° C. The reaction was then stirred for 30 minutes at −78° C., then allowed to warm to room temperature and stir overnight. The mixture was quenched with THF/milliQ H₂O (20 mL, 1:1 v/v). An additional 100 mL milliQ H₂O was added to the resultant mixture which was then extracted into EtOAc (3×70 mL), dried with MgSO₄, filtered and had solvent removed under reduced pressure.

The resulting pale yellow solid was loaded onto a silica gel column with 98 DCM:2EtOH, and eluted with 96 DCM:4 EtOH. Pure fractions were collected and solvent was removed under reduced pressure to leave a pure white powdery solid. Yield: 0.49 g, 73.4%. ¹H NMR (300 MHz, CD₃CN) δ 8.27 (d, J=2.4 Hz, 1H), 8.12 (dd, J=8.4, 2.4 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 4.76-4.61 (m, 4H), 3.55-3.44 (m, 2H). ¹³C NMR (75 MHz, CD₃CN) δ 148.17, 147.59, 141.93, 128.56, 122.89, 122.28, 118.26, 61.50, 61.27. ESI-HRMS for C₈H₉NO₄: [M+Na⁺]⁺: calculated: 206.0424. found: 206.0429.

4-nitrophthalaldehyde

Dry dichloromethane (4 mL) was added to a previously flame dried round bottom flask. The mixture was cooled to −78° C., and oxalyl chloride (195 μl, 2.26 mmol) was added dropwise to the DCM with stirring. Next, dimethyl sulfoxide (250 μl, 3.50 mmol) was added dropwise to the mixture at −78° C., and the mixture was stirred for 15 minutes. The previously prepared (4-nitro-1,2-phenylene)dimethanol dissolved in a DCM/THF mixture (2 mL, 3 DCM:1 THF) was added dropwise and stirred at −78° C. for 2 hours. The flask containing was then rinsed with DCM/THF (2 mL, 3 DCM:1 THF). Neat trimethylamine (2.6 mL, 18.51 mmol) was added to the stirring mixture and then allowed to stir for 10 minutes at −78° C., then allowed to warm to room temperature and then stirred for 30 minutes. The reaction mixture was then quenched with methanol (15 mL), stirred for 10 minutes and solvent was evaporated under reduced pressure yielding a glassy solid. The resulting crude product was purified using a dry loaded column packed with Merck grade silica with 60 Hexanes: 39 EtOAc: 1 MeOH. Pure fractions were collected, pooled and the identity of the compound was confirmed by NMR spectroscopy. Yield: 0.11 g, 52.1%. ¹H NMR (methyl hemiacetal in equilibrium with phthalaldehyde) (300 MHz, CD₃CN) δ 10.53 (s, 1H), 10.48 (s, 1H), 8.71 (d, J=2.3 Hz, 1H), 8.56 (dd, J=8.4, 2.3 Hz, 1H), 8.34-8.11 (m, 7H), 7.61 (m, 3H), 6.60-5.99 (m, 7H), 5.09 (m, 3H), 3.52-3.34 (m, 9H), 3.28 (s, 12H).

3-fluorophthalic acid

Yield: 1.83 g, 62.2%. ¹H NMR (300 MHz, CD₃CN) δ 7.83-7.74 (m, 1H), 7.57 (td, J=8.1, 5.5 Hz, 1H), 7.48-7.39 (m, 1H). ¹³C NMR (75 MHz, CD₃CN) δ 166.15, 165.95, 161.31, 158.03, 132.47, 132.36, 130.71, 130.67, 126.97, 126.93, 124.66, 124.40, 121.30, 121.01, 118.26. ¹⁹F NMR (282 MHz, CD₃CN) δ −117.84 (dd, J=9.3, 5.6 Hz). ESI-HRMS for C₈H₆O₄F: [M+H⁺]⁺: calculated: 185.0245. found: 185.0250.

(3-fluoro-1,2-phenylene)dimethanol

Yield: 0.39 g, 64.4%. ¹H NMR (300 MHz, CD₃CN) δ 7.36-7.27 (m, 1H), 7.23 (d, J=7.1 Hz, 1H), 7.05 (m, 1H), 4.69 (t, J=5.8 Hz, 4H), 3.57 (t, J=5.8 Hz, 1H), 3.43 (t, J=5.8 Hz, 1H). ¹³C NMR (75 MHz, CD₃CN) δ 130.38, 130.25, 125.25, 125.22, 118.26, 115.54, 115.23, 62.89, 62.85, 54.85, 54.76. ¹⁹F NMR (282 MHz, CD₃CN) δ −121.07-−121.30 (m). ESI-HRMS for C₈H₉O₂F: [M+Na⁺]⁺: calculated: 179.0497. found: 179.0484.

3-fluorophthalaldehyde

Yield: 0.14 g, 28.8% (Methyl acetal) ESI-HRMS for C₁₀H₁₁O₃F (methyl acetal): [M+Na⁺]⁺: calculated: 221.0584. found: 221.0590. ¹H NMR (hemiacetal in equilibrium with phthalaldehyde) (300 MHz, CD₃CN) δ 10.46 (s, 1H), 10.31 (s, 1H), 7.79 (dd, J=8.0, 5.1 Hz, 1H), 7.71 (d, J=6.7 Hz, 1H), 7.55-7.40 (m, 20H), 7.28-7.06 (m, 40H), 6.63-5.95 (m, 40H), 4.87 (d, J=20.4 Hz, 20H), 3.76-3.58 (m, 30H), 3.35 (dd, J=14.0, 4.4 Hz, 14H).

4-bromophthalic acid

Yield: 4.43 g, 48.8%. ¹H NMR (300 MHz, CD₃CN) δ 7.88 (d, J=2.0 Hz, 1H), 7.76 (dd, J=8.3, 2.0 Hz, 1H), 7.68 (d, J=8.3 Hz, 1H). ¹³C NMR (75 MHz, CD₃CN) δ 167.94, 167.59, 159.02, 135.16, 135.10, 132.56, 131.85, 131.38, 126.17, 118.26. ESI-HRMS for C₈H₄O₄Br: [M−H⁺]⁻: calculated: 242.9298. found: 242.9293.

(4-bromo-1,2-phenylene)dimethanol

Yield: 0.433 g, 66.5%. ¹H NMR (300 MHz, CD₃CN) δ 7.56 (d, J=2.0 Hz, 1H), 7.43 (dd, J=8.1, 2.0 Hz, 1H), 7.30 (d, J=8.1 Hz, 1H), 4.58 (m, 4H), 3.43 (m, 2H). ¹³C NMR (75 MHz, CD₃CN) δ 143.08, 139.57, 131.18, 130.97, 130.69, 121.56, 118.26, 62.06, 61.97. ESI-HRMS for C₈H₉₀₂Br: [M+Na⁺]⁺: calculated: 238.9678. found: 238.9684.

4-bromophthalaldehyde

Yield: 0.257 g, 52.3%. ¹H NMR (300 MHz, CD₃CN) δ 10.43 (s, 1H), 10.41 (s, 1H), 8.10 (d, J=1.9 Hz, 1H), 8.01-7.97 (m, 1H), 7.88 (d, J=8.2 Hz, 1H). ESI-HRMS for C₉H₉O₃Br (methyl hemiacetal): [M+Na⁺]⁺: calculated: 266.9627. found: 266.9633.

(3-nitro-1,2-phenylene)dimethanol

Yield: 0.90 g, 69.5%. ¹H NMR (300 MHz, CD₃CN) δ 7.70 (m, 2H), 7.47 (t, J=7.9 Hz, 1H), 4.74 (m, 4H), 3.51 (m, 2H). ESI-HRMS for C₈H₁₀O₄N: [M+Na⁺]⁺: calculated: 184.0604. found: 184.0610.

3-nitrophthalaldehyde

Yield: 0.1017 g, 29.4%. ESI-HRMS for C₉H₉NO₅ (methyl hemiacetal): [M+Na⁺]⁺: calculated: 234.0373. found: 234.0378. See table in results and discussion for NMR data.

General HPLC Method

Peptides were purified on an Agilent 1100 series HPLC outfitted with an Agilent Eclipse XDB-C18 column (5 μm internal diameter, 9.4×250 mm) using a linear gradient of solvent A (0.1% Formic Acid H₂O) and solvent B (0.1% Formic Acid MeCN at a flow rate of 2 mL/minute. The column had a void volume of approximately 12 mL (6 minutes.) The gradient went from 0 minutes (85% A, 15% B) to 18 minutes (65% A, 35% B) to 21 minutes (0% A, 100% B) to 26 minutes (0% A, 100% B) to 29 minutes (85% A, 15% B) to 37 minutes (85% A, 15% B.)

Model Isoindoles for Regioselectivity Studies and Photo-Physical Properties

Phthalaldehyde Peptide Quantification Assay⁶⁵

A standard ONB (o-phthalaldehyde-N-acetyl cysteine-borate, 1:1:8) buffer was made using a phthalaldehyde of choice (0.05 M), N-acetyl cysteine (0.05 M), and sodium borate buffer (pH 9.5). This ONB buffer was distributed among 6 vials (3 mL ONB/vial). A standard hexylamine (HAM, 0.007M in MeCN/H₂O, 2:8 in 0.1% formic acid) solution was made and added, increasing incrementally to the 6 vials containing ONB buffer such that a standard curve was made (0-50 uL HAM). These vials were vortexed and then let sit for 30 minutes. Following this, the absorbance of each vial was read at the λ_(max) of each respective isoindole. From these absorbance measurements, a standard curve was formed, from which the extinction coefficient of each phthalaldehyde was determined. In addition, this phthalaldehyde assay was used to determine the concentration of the linear peptides solutions synthesized earlier in the project.

Determining the λ_(max) of Isoindoles

The λ_(max) of each novel isoindole was determined using a wavelength scan on a sample of ONB buffer containing the phthalaldehyde of interest (1.5 mL) reacted with HAM (25 uL). The machine was blanked and a baseline was taken on two ONB samples without HAM added. The spectrometer was used in double beam mode, with one blank ONB sample as a reference.

N-acetyl-S-(2-hexyl-5-nitro-2H-isoindol-1-yl)-L-cysteine

A solution of 4-nitrophthalaldehyde (0.05 M in DMF, 750 μL) was mixed with N-acetyl cysteine (NAc) (0.10 M in DMF, 750 μL) and borate buffer (6 mL, pH 9.5), followed by neat hexylamine (HAM) (25 μL). The mixture was vortexed until it turned a dark orange color. O-phosphoric acid was then added to the reaction mixture until it reached pH 2, upon which it turned a bright orange. The reaction mixture was then extracted into diethyl ether (3×5 mL) and solvent was removed under reduced pressure. The mixture was then column purified on Merck grade silica with 90 DCM:8 EtOH:2 AcOH. Fractions containing the product were collected and solvent was removed under reduced pressure.

General Procedure for Solid Phase Peptide Synthesis

The dried resin contained in a ZEBA Desalt spin column was solvated in 5 mL DMF and shaken for at least 30 minutes. The solvent was drained and the resin was resuspended in 5 mL 2:8 Piperidine/DMF (0.5M Oxyma) and shaken for 5 minutes. The solvent was drained and the resin was resuspended in 5 mL 2:8 Piperidine/DMF (0.5M Oxyma) and shaken for a further 10 minutes. The solvent was drained and the resin was washed with seven 5 mL portions of DMF, with shaking and draining. The resin was then Kaiser tested and if a positive result (purple or brown) was observed, the beads were then resuspended in a 5 mL solution of DMF which already contained 4 equivalents each of Fmoc-Xaa, Oxyma and COMU and ˜11 equivalents DIEA. The resin was then shaken for 1-3 hours at room temperature. The solvent was drained and the resin was washed with five 5 mL portions of DMF, with shaking and draining. The resin was then Kaiser tested and if a negative result (no change in colouration) was observed, the beads were then resuspended in a 5 mL solution of 1:2:2 Ac₂O/Collidine/EtOAc and shaken for 20 minutes at room temperature. If the result of the Kaiser test remained positive, another coupling with same Fmoc amino acid was performed. Once the capping was complete, The solvent was drained and the resin was washed with five 5 mL portions of DMF, with shaking and draining. The resin was then washed with DCM and left to dry under reduced pressure if couplings were complete for the day or resuspended in 2:8 Piperidine/DMF (0.5M Oxyma) for the next Fmoc deprotection.

General Procedure for Peptide Resin Cleavage

To the spin column containing the N-acetylated resin bound peptide was added 5 mL 95:2.5:2.5 v/v/v TFA/TIS/H₂O and the beads were shaken at room temperature for 2 hours. The filtrate was evaporated under reduced pressure to yield a brown or pinkish solid. The last traces of TFA were removed by two successive rounds of co-evaporation with DCM. The crude peptide was then triturated with three 10 mL portions of Et₂O and allowed to air dry overnight to yield an off-white powder. The crude material was purified on a reverse-phase SEP-PACK column (Waters, 3 cc.) Loaded with 0.1% Formic acid H₂O (10 mL,) eluted with successive 10 mL portions of 5:95 MeCN/H₂O (0.1% Formic acid,) 10:90 MeCN/H₂O (0.1% Formic acid,) 15:85 MeCN/H₂O (0.1% Formic acid,) 20:80 MeCN/H₂O (0.1% Formic acid,) and 40:60 MeCN/H₂O (0.1% Formic acid.) Fractions were checked for purity and presence of the product. The purest and most abundant in product fractions where pooled, frozen and lyophilized.

General Fluorescent Isoindole Crosslinking (FLIC) Procedure:

0.5 μmoles of lyophilized linear peptide in the bottom of a 15 mL Falcon Tube was dissolved in 50 μL of Sodium Borate buffer (pH-9.5) and 15 μL of 50 mM ϕ-phthalaldehyde (solution in EtOH) was added at room temperature. The tube was then vortexed at room temperature for 30 seconds. After the appearance of precipitate (5-30 minutes), 35 μL of DMF was added to the reaction mixture to achieve a clear solution. At this point, the entire reaction mixture was injected directly into the HPLC system for immediate purification. The product peak was manually collected, immediately frozen and lyophilized (product tube was wrapped in foil) to yield a white or coloured fluffy powder (28-77% yield).

Loading of 2-CTC Resin with Fmoc-Hyp(OtBu)-OH

1 g (1.2 mmol) of 2-CTC resin was dried against P205 in a desiccator on the high vac line for 4 h. The resin was then transferred to a flame dried round bottom flask under argon and 0.982 g (2.4 mmol, 2 eq) of Fmoc-Hyp(OtBu)-OH was added the flask. The solids were dissolved/suspended in 7.5 mL dry DCM and the reaction mixture was stirred at room temperature. 1.045 mL DIEA (6 mmol, 5 eq) was added dropwise to the stirring solution and the reaction was allowed to proceed over night. 1 mL MeOH was then added and the reaction was stirred for 1 h. The resin was then transferred to a spin column and the resin was thoroughly washed with DCM and dried on vacuum. The solid phase synthesis proceed to the Heptapeptide detailed below as per the general solid phase peptide synthesis procedure outlined in this document.

Synthesis of H₂N-Dap(Boc)-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(OtBu)-OH

The Fmoc protected, resin bound heptapetide was synthesized as detailed above. 100 mg of the resin was then suspended in 5 mL 20% Piperidine in DMF (0.5M) Oxyma and shaken for 5 minutes at room temperature. The solution was drained from the spin column and then 5 mL of fresh 20% Piperidine in DMF (0.5M Oxyma) was added and the solution was shaken for a further 10 minutes at room temperature. The deprotection solution was then drained and the resin was resuspended in DMF, shaken and drained. This washing procedure was repeated a further 6 times. The resin was then washed with DCM and Kaiser tested. Upon confirmation of the presence of free amine, the resin was resuspended in 5 mL of a 4:1 v/v solution of DCM/HFIP and shaken at room temperature for 20 minutes. The solution was then drained into a round bottom flask and the resin was washed with DCM and these rinses were pooled with the original filtrate. The solution was then evaporated under reduced pressure to yield a pinkish beige solid which was used in the next step with no further purification. (42.3 mg, 32.5 μmol). HRMS (m/z) [M+H]⁺ calcd. for C₇₂H₈₈N₉O₁₂S, 1302.6273; found 1302.6376.

Synthesis of (2S,3R,4R)—Oγ,Oδ-bis-TBS-Nα-Fmoc-dihydroxyisoleucine-NHS

Reagents and conditions: a) E-Butene, dry tBuOK, BuLi, dry THF, −78° C. then (+)-(Ipc)₂BOMe then BF₃-Et₂O, then Benzyloxy-acetaldehyde −78° C., 4 h, then NaOH, H₂O₂, reflux, 1 h. 92% yield. b) TBDMSOTf, 2,6-Lutidine, DCM, 4° C. to rt, 6 h. 48% yield. c) OsO₄, NMO, Acetone/H₂O rt, O.N. 54% yield. d) NaIO₄, MeOH/H₂O, rt, 1 h. 73% yield. e) S-Phenylethylamine, KCN, MeOH/H₂O rt, 3 days. 39% yield. f) 6MHCl, reflux, 6 h. 66% yield. g) H₂, MeOH, rt O.N. h) LiOH, H₂O, rt, 4.5 h. i) TBDMSCl, Imidazole, dry DMA, rt O.N. j) Fmoc-Osu, Na₂CO₃, 1,4-dioxane/H₂O. 22% overall yield over 4 steps. k) DSC, Collidine, EtOAc/MeCN, 4° C. to rt, 6 hours. 70% yield.

All spectra matched a previous literature report³⁸.

Synthesis of H₂N-DhIle(Bis-O-TBS)-Dap(Boc)-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(OtBu)-OH

i) H₂N-Dap(Boc)-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(OtBu)-OH (42.3 mg, 32.5 μmol) was dissolved in 87 μL DMF in 2 mL flat bottom vial and 14.1 μL DIEA (8.1 μmol, 2.5 eq) was to added to solution along with 34.8 mg (2S,3R,4R)—Oγ,Oδ-bis-TBS-Nα-Fmoc-dihydroxyisoleucine-NHS (48.8 μmol, 1.5 eq.) The mixture was stirred overnight at room temperature.

ii) Upon completion of the acylation, 20 μL Et₂NH was added to solution and the reaction was stirred for 2 h at room temperature. The volatiles were then evaporated and the crude was placed on the high vac line overnight.

iii) The resulting gel was dissolved in 600 μL DMF and 200 μL 1.0M TBAF in THE solution was added, the resulting solution was stirred at room temperature for 2 and a half hours. Upon completion of the deprotection (as assessed by LRMS-ESI) the reaction mixture was diluted with 10 mL 1:1 H₂O/MeCN (0.1% Formic Acid), shell frozen and lyophilized. The resulting gel was purified on Reverse-Phase SEP-PACK, using gradient elution from 7:3 H₂O/MeCN (0.1% Formic Acid) to 2:8 H₂O/MeCN (0.1% Formic Acid.) The fractions containing the product were pooled, frozen and lyophilized to yield a fluffy white powder. (22.4 mg, 15.5 μmol 48% yield over 3 steps.). HRMS (m/z) [M+H]⁺ calcd. for C₇₈H₉₉N₁₀O₁₅S, 1447.7012; found 1447.7018.

Macrolactamization and Global Deprotection of H₂N-DhIle(Bis-O-TBS)-Dap(Boc)-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(OtBu)-OH

i) H₂N-DhIle(Bis-O-TBS)-Dap(Boc)-Gly-Ile-Gly-Cys(Trt)-Asn(Trt)-Hyp(OtBu)-OH (22.4 mg, 15.5 μmol) was dissolved in 4 mL DMA, 53 mg HATU (139 μmol, 9 eq) was added to the reaction mixture along with 26.9 μL DIEA (155 μmol 10 eq) and the solution was stirred at room temperature for 1 h. Upon completion of the reaction (as assessed by LRMS-ESI) the reaction mixture was diluted with 20 mL H₂O, shell frozen and lyophillized.

ii) Half of the resulting solid was dissolved in 1 mL 95:2.5:2.5 TFA/TIS/H₂O in a round bottom flask and stirred for 1 h at room temperature. The volatiles were evaporated under reduced pressure and the resulting solid was tritured with one cold 10 mL portion of Et₂O. The resulting solid was purified Reverse-Phase SEP-PACK, using gradient elution from 1:0 H₂O/MeCN (0.1% Formic Acid) to 7:3 H₂O/MeCN (0.1% Formic Acid.) The fractions containing the product were pooled, frozen and lyophilized to yield a fluffy white powder. (Quantitative yield over 2 steps, contaminated with HATU). HRMS (m/z) [M+H]⁺ calcd. for C₃₁H₅₃N₁₀O₁₂S, 789.3565; found 789.3567.

Condensation of cyclo-[DhIle-Dap-Gly-Ile-Gly-Cys-Asn-Hyp] with o-phthalaldehyde

0.5 μmoles of lyophilized monocyclic peptide in the bottom of a 15 mL Falcon Tube was dissolved in 80 μL of 50 mM TCEP in Sodium Borate buffer (pH-8) and 15 μL of 50 mM o-phthalaldehyde (solution in EtOH) was added at room temperature. The TCEP was added to ensure the cysteine remained in the free thiol form, as disulfide formation in the precursor was observed over time. The tube was then vortexed at room temperature for 30 seconds. Allowed to react for a further 45 minutes, after which a clear yellow solution was observed. At this point, the entire reaction mixture was injected directly into the HPLC system for immediate purification using Method D. The product peak was manually collected, immediately frozen and lyophilized (product tube was wrapped in foil) to yield a white powder (33% yield by area under the curve at 335 nm). HRMS (m/z) [M+H]⁺ calcd. for C₃₉H₅₄N₁₀O₁₂SNa, 909.3541; found 909.3538.

FlICk Amanitin Modelling

A mol file of both FlICk amanitin variants was imported into Avagadro. Due to lack of 3D information in the file, the resulting structures were manipulated to roughly mimic the crystal structure of β-amanitin (the crosslink was pulled onto the same face of the macrocycle as in all known amatoxins, amides where made trans, etc.) The structures were then energy minimized using the MMF94s method with steepest decent gradient. The resulting structural models were imported into Mercury and images were captured.

FlICk Amanitin MTT Assay

Both FlICk amanitin and authentic α-amanitin (purchased from Sigma Aldrich) were dissolved in H₂O. The FlICk amanitin concentrations used were 100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM, 0.00128 μM. The α-amanitin concentrations used were 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, 0.0032 μM, 0.00064 μM, 0.000128 μM, 0.0000256 μM. The cell culture and MTT assay procedures were carried out as per our report on the total synthesis of α-amanitin³⁸. The MTT assays were run in triplicate. The IC50 assay curves were produced using Prism Software.

EXAMPLES

Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

Example 1: Melanocortin Analogs

We began our investigation of FlICk induction using commercially available OPA that was deployed in the cyclization of four linear α-MSH analogues based on the monocyclic Melanotan-II sequence. Notably, Melanotan-II [N-Acetyl-L-norleucyl-L-α-aspartyl-L-histidyl-D-phenylalanyl-L-arginyl-L-tryptophyl-L-lysinamide (2→7)-lactam], a very potent (K_(d)˜1 nM) macrocyclic heptapeptide MSH analog²⁸, has been clinically trialed for erectile dysfunction²⁹ and is used illicitly to promote tanning. Nevertheless, apart from Melanotan-II, most monocyclic heptapeptide MSH analogs typically show much higher K_(d) values (K_(d)>20 nM), which not only makes Melanotan-II unique among MSH analogs but underscores the synthetic challenges associated with generating high affinity heptapeptide MSH analogs³⁰. Hence, four heptapeptides were synthesized with increasing length of the amine-bearing side chain:amino proprionic acid, amino-butyric acid, ornithine, and lysine (FIG. 2 ) to assess the effect of ring size in a competitive binding assay against Melanocortin Receptor 1 (MC1-R.)

Operationally, the reaction is especially user-friendly. The peptide, in the form of a lyophilized powder, is dissolved in borate buffer (pH 8), to which the o-phthalaldehyde, often isolated as a methyl-hemiacetal (see ESI) is added in EtOH along with a small amount of DMF to aid solubility. The combined solution is vortexed and the reaction mixture is directly subjected to resolution by HPLC. With 2 eq. of OPA at 10 mM, the reaction progress of FlICk induction was too fast to be accurately measured and the reaction was judged to be complete within 30 sec. The entire mixture is then loaded onto a standard C-18 SepPak or HPLC without further manipulation and the product peak is collected, frozen, and lyophilized. Peak-to-peak conversion was observed by HPLC (see supporting information) with yields ranging from (54-75%, beginning with linear peptides that were between 75 and 80% pure). Impressively, three of four FlICked MSH derivatives showed high affinity for MC1-R (Table 1). Evident from the results is the apparent need for a minimum ring size to achieve binding affinity comparable to the native α-MSH (IC₅₀=2.6 nM)³¹. One-carbon homologation of the DAP variant of the peptide results in an order of magnitude increase in binding affinity, while there appears to be an ideal ring size comprising ornithine as lysine substitution moderately diminishes binding affinity.

TABLE 1 Data for MSH derivatives prepared according to FIG. 2. MSH (CH₂)_(n) Yield % K_(i) A n = 1 75  35 nM B n = 2 58 2.5 nM C n = 3 64 1.7 nM D n = 4 54 4.0 nM

To access modified isoindoles with an eye to varying the absorbance and emission wavelengths, we synthesized five modified ortho-phthaldehydes (FIG. 3 ) by readily-accessible two- or three-step synthetic routes starting either with the corresponding xylene or phthalic acid (see ESI).

With these OPA derivatives in hand, we characterized the photophysical properties of the isoindoles that form quantitatively. Using the standard phthalaldehyde assay conditions for quantifying an amine via we added a known concentration of n-hexyl-amine (7 mM) and excess of OPA and NAc-cysteine to generate a known quantity of isoindole. Reaction progress was monitored by UV-vis spectroscopy; with the exception of the NOPAs, the reaction was judged to be complete within 30 seconds. Absorbance spectra were recorded and apparent molar extinction coefficients were calculated and compared to those of commercially available OPA, used as a standard, for which the quantum yield is known to be 0.47. Quantum yields were then measured accordingly (see ESI for detailed procedure). Results are reported in Table 2 in the context of FlICked MSH derivatives.

TABLE 2 Photophysical properties of modified isoindoles and K_(i) values for corresponding MSHs constructed with modified OPAs. For entry-1, two regio-isomers (a and b) were separated and were found to have very similar K_(i) values. Stokes Entry OPA λ_(ex.) λ_(em.) Shift Φ K_(i) 1 3-FOPA 329 nm 415 nm  86 nm 0.04 a: 2.2 nM b: 1.7 nM 2 4-FOPA 338 nm 454 nm 116 nm 0.31 1.2 nM 3 3-NOPA 487 nm NA NA <0.001 3.0 nM 4 4-NOPA 444 nm NA NA <0.001 1.5 nM 5 4-BrOPA 345 nm 452 nm 116 nm <0.001 1.6 nM Since MSH-C (FIG. 2 ) showed the highest affinity, the same linear precursor was condensed with these 5 new OPAs. FlICked MSH derivatives prepared from both FOPAs and the BrOPA formed within 30 seconds while those prepared from both NOPAs required approximately 15 min (reaction progress was monitored by recording the corresponding isoindole absorbance vs time). As expected, all five FlICked MSH derivatives showed similar Ki values (Table 1). FlICked MSH derivatives were found to be stable for over one month when stored at −20 deg. On standing in aqueous conditions, a small amount of unknown oxidized product (M+16)⁺ was observed by ESI and was taken to likely resulted from reaction with molecular oxygen on the isoindole ring³². In addition, extended irradiation of the unmodified isoindole with UV-light or ambient sunlight led to observable photobleaching. By contrast, the electron-withdrawing substituents (F, NO₂) on the isoindole increase photostability and shelf-life, with the nitro derivatives greatly reducing unwanted oxidation at room temperature in aqueous solution (as observed by ESI mass spec.).

FlICked MSHs with fluoro-isoindoles exhibit vivid blue-green fluorescence (see graphical abstract) with spectral properties and quantum yields that are comparable to those of blue fluorescent proteins (BFPs).³³ While we had hoped that fluorescence could be exploited for visualizing MC1-R binding and internalization, the well-known background fluorescence due the over-production of melanin in melanocytes unfortunately impeded this application (data not shown).

Not surprisingly, isoindole formation on 3- and 4-substituted OPAs occurs with varying degrees of regioselectivity (see ESI) and, in certain cases the FlICked products were difficult to separate by standard reverse-phase HPLC. Nevertheless, in the case of the FlICk-MSH formed from 3-FOPA (Table 2, entry 1), two regio-isomeric FlICk products were readily separated in approximately 3:4 ratio based on the UV-absorbance at 280 nm (Trp absorbance). Both regioisomers show A_(280 nm)/A_(335 nm) ratios that are within experimental error (˜5%) thus demonstrating that both isoindoles have nearly identical molar extinction coefficients. In addition, each regioisomer shows nearly identical binding affinities (Table 2, entries 1a and 1b). While the apparent inseparability of these regioisomers may reflect a current limitation of this method, a similar limitation has arisen in certain instances where cis/trans alkene-stapled peptides are obtained as inseparable mixtures of cis/trans isomers^(12c, 12d). Notwithstanding such inconvenience, metal-mediated cross-metastasis is extensively pursued for stapling in biotechnology and medicine.

To expand the scope of this reaction, we reacted the other three linear MSH precursors (FIG. 1 , where n=1,2,4) with each of these OPAs to afford a focused library of HPLC-purified 20 MSHs (Table 3 and ESI for characterization). While affinities were not measured at this juncture, we were able to produce all 20 HPLC-purified peptides over the course of just two days, a feature that highlights both the facility and robustness of this reaction for use in applications of directed library development.

TABLE 3 Summary of FlICK MSH library. Yields are calculated by LC at 280 nm (tryptophan absorbance) as entire reaction mixture was directly subject to HPLC. (CH₂)_(n) 3-FOPA 4-FOPA 3-NOPA 4-NOPA 4-BrOPA n = 1 73%* 74%  66%* 47% 49% n = 2 60%  55% 71% 62% 39% n = 3 77%* 59% 35% 36% 36% n = 4 65%* 53% 39% 28% 29% Asterisks indicate combined yields of HPLC-separable regioisomers.

Example 2: Amatoxin Analogs

Given this preliminary success in FlICking a linear precursor to achieve a high-affinity monocyclic MSH analog, we applied this strategy to produce a challenging bicyclic peptide that was designed to mimic α-amanitin, a well-known cytotoxin. Both α- and β-amanitin (FIG. 4 ) possess a characteristic hydroxy-tryptathionine sulfoxide crosslink that rigidifies the macrocycle thereby restricting key orientations of the side chains which engage in significant interactions (H-bonding, hydrophobic) with the bridge helix of RNA Pol II. The indole of the tryptathionine crosslink engages in a 7-cation interaction with arginine-726 (A chain of RNA Polymerase) along with other hydrophobic interactions.³⁴ Whereas α-amanitin is cytotoxic to CHO cells (IC₅₀˜0.5 μM), the synthetically accessible didexoy-derivative that lacks both the (R)-sulfoxide and the 6-hydroxy group on the tryptathionine is slightly less cytotoxic to CHO cells (IC₅₀˜2 μM)³⁵.

We hypothesized that an isoindole, formed from DAP and cysteine, would serve as a fluorescent bio-isostere to replace the tryptathionine in α-amanitin, while retaining the same general connectivity in terms of the intra-annular distance between the two Ca carbons. In considering a FlICked amanitin analog, two possible isoindole orientations are conceivable given the circularly permutable nature of the peptide macrocycle as shown below in FIG. 4 .

Molecular modelling and energy minimization on FlICked amanitin-1& -2 suggested that FlICk-amanitin-1 would adopt a structure that would be similar to β-amanitin for which a crystal structure is known³⁶ (note α-amanitin adopts a similar solution structure³⁷). When compared with a crystal structure of β-amanitin, the gross structural features of the FlICk-amanitin-1 model appear similar. The tilt-angle of the indole relative to the macrolactam ring is similar whereby both indolyl crosslinks lie over the eastern ring of the molecule. Furthermore, the amide bonds appear to adopt a similar orientation, most notably in terms of the spatial arrangement of the cysteine NH, the asparagine NH and the carbonyl of Gly⁵ which are suggested to be participating in hydrogen bonding per the modelling study. The most notable difference may be in the orientation on the amide bond between dihydroxyisoleucine and tryptophan/DAP and the orientation of the aspartate/asparagine side chain. By contrast, the modelled structure of FlICk-amanitin-2 appears grossly different from the crystal structure of β-amanitin; the isoindole now lies directly over the western ring of the molecule, veering out towards the viewer while the backbone structure appears contorted, particularly in the eastern j-turn.

Guided by this preliminary modelling, we readily synthesized FlICk-amanitin-1 in accord with the scheme shown in FIG. 5 . Using a solid-phase synthesis strategy on 2-CTC resin with acid-labile side chain protection, the heptapeptide containing diaminoproprionic acids was synthesized on the solid phase. Following cleavage from the resin with mild acid (leaving the side chain functional groups concealed), the unnatural amino acid, dihydroxyisoleucine³⁸ was coupled to the heptapeptide in solution, which was then deprotected and cyclized with HATU in DMA. TFA treatment yielded a macrocycle, with the amine and thiol functionalities, primed for FlICk cyclization that was completed essentially upon mixing.

Whereas the monocyclic octapeptide precursor showed no detectable toxicity (data not shown), FlICk-amanitin-1 showed detectable toxicity at a level that is approximately 100-fold less toxic than that of α-amanitin (FIG. 6 ). Critically, FlICk-amanitin-1 is only 20-fold less toxic than dideoxy-amanitin (IC₅₀ of 2 μM), its more closely related congener. The diminished toxicity of FlICk-amanitin-1 may be understood in terms of a different orientation of the isoindole compared to the tryptathionine. Subtler differences in the intra-annular distances may also explain the reduced toxicity. Nevertheless, assuming that toxicity directly correlates with affinity for Pol II, a 20-fold difference in toxicity reflects less than 2 kcal/mol difference in binding energy. It is conceivable that further modification of the isoindole along with side-chain modifications may eventually provide for synthetic toxins of near-native toxicity. For the first time, a bioactive amanitin analog has been synthesized whereby the tryptathionine is replaced with a bio-orthogonal isoindole. This result now demonstrates that bicyclic peptides may be facilely re-engineered to with novel crosslinks for added bioactivity.

In conclusion, we have developed a methodology for rapidly accessing cross-linked fluorescent mono- and bicyclic peptides via the formation of a fluorescent isoindole crosslink. In so doing, we also developed a route to modified o-pthalaldehydes and measured the photophysical properties of the isoindoles they engender. We have applied this strategy to a clinically relevant peptide, α-MSH, and produced several high-affinity ligands with low single-digit nanomolar binding affinities. We then applied this strategy to a monocyclic peptide of no cytotoxicity to create a FlICked-bicycle that shows cytotoxicity and, which based on molecular modelling, structurally mimics α-amanitin, a highly toxic and medicinally relevant bicyclic peptide natural product. Taken together, these results demonstrate that this method may be a reliably augment bioactivity from inactive precursors. The utility of this approach lies in its high chemoselectivity, mild conditions, and operational simplicity. Someone skilled in the art may anticipate that OPAs could be further derivatized with azides, alkynes, biotin, or other reporter groups to increase the chemical functionality of FlICked peptides. Interestingly, FlICked peptides prepared from the FOPAs show vivid fluorescence blue-green with reasonable high quantum yields reminiscent of BFPs. We expect that the fluorescent nature of these peptides may enhance their value as probes in cell biology and for screening applications with built-in fluorescence. These findings portend success in applying FlICk synthesis to enhance the chance of discovering new bioactive mono- and bicyclic peptides from combinatorial arrays³⁹ and may possibly find additional utility for identifying cyclic peptides via phage display⁴⁰ and ribosome-display^(16h), and for generating chemically constrained fluorescent proteins and antibody fragments.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. Titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.

REFERENCES

The entire disclosures of all applications, patents, and publications, cited above and below, are hereby incorporated by reference. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in Canada or any other country.

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What is claimed is:
 1. A compound comprising a cyclic peptide of Formula IIb wherein: Formula IIb has the structure:

wherein: R¹ is —Ac or —H; R² is -Nle-; X¹ is -His- or -D-His-; X² is -Phe- or -D-Phe-; X³ is -Arg- or -D-Arg-; X⁴ is -Trp- or -D-Trp-; A¹, A², A³, A⁴ and A⁵, are each independently, hydrogen, halogen, NO₂, CN, alkyne, azide, aryl, heteroaryl, COOH, SO₃ ⁻, CF₃, RCO, CONHR, NH₂, NHR, NR₂, ether, thioether, hydroxyl, or boronate; R is an alkyl or an alkenyl, each of which is optionally substituted; B¹, B², B³, B⁴ and B⁵, are each independently, carbon or nitrogen; and x and y, are each independently, a natural number between 1-15. 